The primary aim of the proposed research is to investigate the role of the epidermal growth factor receptor (EGFr) and transforming growth factor type alpha (TGFalpha) during multi-stage skin carcinogenesis, and in particular, during the process of skin tumor promotion. A variety of studies, primarily using cells in culture, have shown that phorbol esters and other types of tumor promoters initially lead to reduced binding of EGF to its receptor. However, recent studies from our laboratory have demonstrated that topical applications of both phorbol ester, as well as, anthrone type tumor promoters to mouse skin in vivo, after an initial decrease, ultimately lead to significant elevations of EGF binding to its receptor. These elevations in EGF binding occurred at times when epidermal protein kinase C (PKC) was significantly downregulated by both promoters. Treatment with both types of tumor promoters also leads to increased amounts of EGFr protein and to increased expression of TGFalpha species. The Specific Aims of this proposal are: 1. To determine the effects of topical treatment with phorbol esters and anthrones on the level and tyrosine kinase activity of the mouse keratinocyte EGFr; 2. To determine the ability of tyrphostins (tyrosine kinase inhibitors) to inhibit the keratinocyte EGFr tyrosine kinase activity, epidermal cell proliferation and tumor promotion by phorbol esters and anthrones; 3. To determine the effects of topical treatment with phorbol esters and anthrones on the phosphorylation of Thr-654, 669, and possibly other Thr or Ser residues of the EGFr; to determine which epidermal PKC isozymes phosphorylate the keratinocyte EGFr; and to determine whether promoter-induced loss of specific PKC isozyme correlates with the alterations in EGFr phosphorylation state; 4. To further explore the nature of, mechanism(s) for, and functional significance of altered expression of mouse epidermal TGFalpha species in promoter treated vs. control epidermis. As part of this Specific Aim, we will generate and utilize transgenic mice, with the TGFalpha gene targeted to the skin using either the bovine keratin 5 or 10 promoters to examine the functional significance of elevated TGFalpha levels; and 5. To examine the relationship between changes in the expression of the EGFr and TGFalpha and the progression of premalignant to malignant skin lesions. These studies will examine EGFr/TGFalpha expression in both early and late stage papillomas and in carcinomas and, in collaboration with Project 1 will attempt to define the relationships between genotypic changes occurring during tumor progression and phenotypic changes involving altered expression of EGFr/TGFalpha. This experimental approach will allow us to test the hypothesis that repeated topical application of phorbol ester and anthrone tumor promoters leads to sustained loss of a negative feedback mechanism involving phosphorylation at Thr-654 (and possibly other sites) of the EGFr by PKC and that the concomitant elevation of the EGFr and/or ligands, such as TGFalpha, may provide a mechanism for sustained cell proliferation essential for skin tumor promotion. Furthermore, alterations in EGFr and TGFalpha expression may play a critical role in the acquisition of promoter-independent growth and ultimately in the progression of skin tumors from the premalignant to malignant state.